Figure 4. FzlA affects FtsZ structure in vivo and promotes formation of higher order FtsZ structures in vitro.

A) Localization of FtsZ-CFP in cells from FzlA depletion strain EG312 grown in xylose (fzlA induced) or glucose (FzlA depleted) for 24 h. Arrows indicate morphologically normal FtsZ rings. B) Localization of FtsZ-CFP in Caulobacter bearing a plasmid with P_xylX driving overexpression of fzlA. Cells were grown for 23 h in glucose (uninduced) or xylose (FzlA overproduced). Arrows indicate FtsZ-CFP foci associated with the sides of the cell. Hatched arrows indicate multiple mislocalized FtsZ-CFP foci. In A-B, fluorescence (red) overlaid on phase contrast (PC) (left) and fluorescence (right) are shown. Scale bars = 2 μm. C) (Left) Coomassiestained SDS PAGE of pellet fractions after high speed centrifugation of FtsZ polymerization reactions containing the indicated concentrations of FzlA and 2 μM FtsZ. (Right) Bar graph depicting relative amount of FtsZ in the pellet at each FzlA concentration. Graph is normalized relative to FtsZ in the pellet with 0 μM FzlA and mean and SEM are shown (n = 3). D) Coomassie-stained SDS PAGE of pellet fractions after low speed centrifugation of FtsZ polymerization reactions containing 2 μM FtsZ ± 4 μM FzlA. E) Right angle light scattering over time of FtsZ in the presence of the indicated concentrations of FzlA. Arrow indicates addition of FzlA or buffer control and hatched arrow indicates addition GTP. F) Right angle light scattering reactions as in E, but containing 0.1 mM of the indicated nucleotide. 4 μM FzlA is included where indicated. Inset graph is an expanded view of the bottom of the graph at left. G) Inorganic phosphate (P_i) concentration in solution over time in the presence of 2 μM FtsZ, 2 mM GTP, and the indicated concentrations of FzlA. GTPase rates (P_i released per FtsZ molecule per min) for each reaction are indicated. See also Fig. S3.